Dyes and Pigments 99 (2013) 588e593

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Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig

Binding of sulforhodamine B to human serum albumin: A
spectroscopic study
Masanori Kitamura a, Koji Murakami a, Kohei Yamada a, Keiichi Kawai b,
Munetaka Kunishima a, *aFaculty of Pharmaceutical Sciences, Institute of Medical, Pharmaceutical, and Health Sciences, Kanazawa University, Kakuma-machi, Kanazawa 920-1192,
Japan
Faculty of Health Sciences, Institute of Medical, Pharmaceutical, and Health Sciences, Kanazawa University, 5-11-80 Kodatsuno, Kanazawa 920-0942,
Japanba r t i c l e i n f o

a b s t r a c t

Article history:
Received 6 March 2013
Received in revised form
20 April 2013
Accepted 13 June 2013
Available online 22 June 2013

Sulforhodamine B (SRB) is a widely used ﬂuorescent dye in biological sciences including in vivo studies.
In this study, the binding interaction between SRB and human serum albumin (HSA) was investigated by
ﬂuorescence and circular dichroism spectroscopy. Complexation with HSA leads to an increase in dye
ﬂuorescence, from which the binding constant (1.6  105 M1 at 25  C), binding stoichiometry (1:1), andthermodynamic parameters of the SRBeHSA interaction (DH ¼ 5.2 kJ/mol, DS ¼ 116.5 J/mol K) wereobtained. The large positive DS and the small positive DH indicate an entropy-driven binding process,suggesting that SRB binds to Sudlow site I of HSA by a dominant hydrophobic interaction. Circular dichroism revealed some degree of conformational changes in HSA upon binding.
Ó 2013 Elsevier Ltd. All rights reserved.

Keywords:
Sulforhodamine B
Human serum albumin
Fluorescence
Fluorescence quenching
Circular dichroism
Albumin

1. Introduction
Sulforhodamine B (SRB) shown in Chart 1 has advantageous
spectral characteristics, such as high molar extinction coefﬁcient[1], high ﬂuorescence quantum yield [1,2], pH-independent emission [3,4], and narrow-band laser performance [5]. In addition, SRB
also has considerable photostability [5,6], high water solubility [7],and low biological toxicity [8]. Therefore, it has been extensively
used as a dye during the past few decades in laser technology [5,6],
the food coloring industry [9,10], and especially in biological sciences [7,11e23]. The SRB assay is a well-known method to measuredrug-induced cytotoxicity and cell proliferation in vitro for largescale screening applications [11e13]. SRB has also been applied to
study cellecell communications [14] and neuronal morphology[15,16] and to model hydrophilic drugs in vitro [7,17e19]. Recently,
the in vivo imaging of elastic and collagen ﬁbers was developed
using SRB and two-photon excitation microscopy [20]. This technique allows the noninvasive visualization of living tissue at highdepths, because near infrared light is only weakly absorbed by* Corresponding author. Tel./fax: þ81 76 264 6201.
E-mail address: kunisima@p.kanazawa-u.ac.jp (M. Kunishima).
0143-7208/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.dyepig.2013.06.011

tissue and water (e.g., the optical window in biological tissue).
Interestingly, SRB can cross the bloodebrain barrier after intravenous injection, and the two-photon excitation method using SRB
was also exploited for the imaging of astrocyte cells in the brains ofmice [21e23]. Because of the rapid increase in SRB usage, it ispossible that SRB will be used for the ﬂuorescence imaging of thehuman body. Hence, it is essential to understand the interaction of
SRB with human serum albumin (HSA) in the blood.
HSA is the most abundant protein in plasma, which consists ofapproximately half of the proteins found in human blood, with aconcentration of approximately 6.0  104 M [24]. One of its functions
is to carry various exo- and endogenous compounds, such as fatty
acids, bilirubin, tryptophan, steroids, and drugs, via two major bindingsites in subdomains IIA and IIIA, namely Sudlow sites I and II, respectively. The fact that HSA can bind to dyes is evidenced by the numerous
studies of the binding of various organic dyes with HSA [25e29].
However, one study concluded that SRB does not bind to the plasma
albumin of mice, but experimental details were not provided [20]. In
this context, it is worth studying the binding between SRB and HSA.
In this study, we report the experimental study of the binding of
SRB with HSA at physiological pH using steady-state ﬂuorescenceand circular dichroism spectroscopy. The binding parameters, theM. Kitamura et al. / Dyes and Pigments 99 (2013) 588e593

Et

Et
N

Et
N

O

Et

SO3

SO3
Chart 1. Structure of SRB.

identiﬁcation of binding sites, and the nature of forces in theinteraction will be beneﬁcial for the use of SRB in the food coloring
industry and in diagnostic imaging.

589

molecular distances. The ﬂuorescence intensity of SRB is sensitive toenvironmental solvent polarity [2,33]. For example, the ﬂuorescencequantum yield of SRB in water (F ¼ 0.39) is smaller than that in a
mixture of water and dioxane (F ¼ 0.79 where water/dioxane ¼ 2:98)[2]. 1-Anilinonaphthalene-6-sulfonic acid (ANS) is weakly or nonﬂuorescent in water but strongly ﬂuoresces when bound to HSA or
bovine serum albumin (BSA) [34]. As was observed in ANS, the ﬂuorescence intensity of SRB also increased upon the addition of HSA(Fig. 1), indicating microenvironmental changes around SRB and theinclusion complexation of SRB by HSA. As shown in Fig. 2, thecontinuous variation method (Job’s plot) using ﬂuorescence from SRB
suggests 1:1 inclusion complexation stoichiometry between SRB and
HSA [35,36]. This ﬂuorescence spectral change of SRB in the presenceof HSA was also analyzed by the Scatchard equation (2):r=Df ¼ nK  rK;(2)

2. Experimental
2.1. Materials
Human serum albumin (free of fatty acids) was purchased from
Seracare Life Sciences, and sulforhodamine B (C.I. Acid Red 52, laser
grade, 95%) was obtained from Aldrich, and both were used without
further puriﬁcation. Deionized water was generated by an Elix UV
System from Millipore.

3.2. Binding mode

Absorption and circular dichroism spectra were recorded on a
Shimadzu UV-2400PC spectrophotometer and JASCO J-820 spectrometer, respectively. Fluorescence spectra were measured on a
JASCO FP-6500 spectroﬂuorometer equipped with an ETC-273T
thermostatic cell holder using a 1.0 cm path length quartz cell.
2.3. Procedures
2.3.1. Inner-ﬁlter effect of SRB during the measurement of
ﬂuorescence from HSA
When the ﬂuorescence spectra of HSA were measured (excitation wavelength ¼ 295 nm, emission wavelength ¼ 340 nm) for the
SterneVolmer plot, the intensities were corrected for the absorption of the exciting light and the reabsorption of the emitted light todecrease the inner-ﬁlter effect by using equation (1) [30e32].

(1)

where Fcorrected is the corrected ﬂuorescence value, Fobserved is themeasured ﬂuorescence value, Aex and Aem are the absorbance of thesystem at the excitation wavelength (295 nm) and at the emission
wavelength (340 nm), respectively.
2.3.2. Binding studies between HSA and SRB
Stock solutions of SRB and HSA were prepared with suitableconcentrations for each experiment. The interactions between SRB
and HSA were monitored by ﬂuorescence and circular dichroism
spectroscopic techniques.
3. Results and discussion
3.1. Fluorescence characteristics of SRB upon complexation with
HSA
Fluorescence measurements can provide some information on thebinding of small molecules to proteins, such as binding constants,
binding sites, binding modes, binding mechanisms, and interIf the enthalpy change (DH) does not vary signiﬁcantly in thetemperature range, both enthalpy change and entropy change (DS)can be calculated from the van ’t Hoff equation (3):ln K ¼ DH=RT þ DS=R;(3)

where K is the binding constant at the corresponding temperature,
and R is the gas constant. The DG, DH, and DS values for SRBeHSA
complexation are obtained from Fig. 4 and are listed in Table 1.
There are several types of non-covalent interaction modes between a small molecule and a protein, including hydrogen bonding,electrostatic interaction, van der Waals force, and hydrophobic
interaction. The binding modes of Sudlow site I with small molecules are mainly by hydrophobic interactions, whereas those of
Sudlow site II involve a combination of hydrophobic, hydrogenbonding, and electrostatic interactions [37,38]. A positive DS andinsigniﬁcant DH value frequently suggest hydrophobic interaction,

λmax = 582 nm

1000
Fluorescence intensity (a. u.)

2.2. Instrumentation


Fcorrected ¼ Fobserved  antilog½ðAex þ Aem Þ 2;

where r represents the number of moles of bound SRB per mole of
HSA, Df shows the molar concentration of free SRB, n and K are thenumber of binding sites and the binding constant, respectively.
From the Scatchard plot of the SRBeHSA system at 25  C (Fig. 3), thebinding constant between SRB and HSA was estimated as
1.6  105 M1. Table 1 summarizes the binding constants at various
temperatures (15e35  C).

(e)

800
600

(a)
400
200
0
500

550
600
Wavelength (nm)650

Fig. 1. Fluorescence spectral change of SRB (5 mM) in the presence of HSA (0e16 mM) in
phosphate buffer (67 mM, pH 7.4) at 25  C (excitation at 425 nm). The HSA
concentrations were (a) 0, (b) 4, (c) 8, (d) 12, and (e) 16 mM.

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M. Kitamura et al. / Dyes and Pigments 99 (2013) 588e593
Table 1
Binding constants obtained from Scatchard plots and thermodynamic parameters of
the SRBeHSA interaction at pH 7.4.

F−F0 (a. u.)

300

200

T (K)

log K

n

DG (kJ/mol)

DH (kJ/mol)

DS (J/mol K)

288
298
308

5.1
5.2
5.2

1.03
1.05
1.00

28.3
29.7
30.7

5.2

116.5

F0 =F ¼ 1 þ Ksv ½Q ;

100

0
0

0.2

0.4
0.6
[SRB]/([SRB + HSA])

0.8

1

Fig. 2. Job’s plot of SRB bound to HSA in phosphate buffer (67 mM, pH 7.4) at 25  C(excitation at 425 nm, emission at 625 nm). [SRB þ HSA] ¼ 40 mM. F 0 and F are theﬂuorescence intensities of SRB in the absence and presence of HSA, respectively.

where water molecules arranged in an orderly manner around asmall molecule and a protein are released and are disordered [39].
The large positive DS and the small positive DH shown in Table 1
indicate an entropy-driven binding process of amphiphilic SRB[40], implying that the binding of SRB to Sudlow site I of HSA issimilar to that of warfarin [41,42] and azidothymidine (AZT) [43] tothe site I and exhibits similar thermodynamic parameters.
3.3. Fluorescence quenching of HSA
Fluorescence quenching refers to the decrease in the ﬂuorescence quantum yield of a ﬂuorophore due to various molecularinteractions, including excited-state interactions, molecular rearrangements, energy transfer, groundestate complex formation, andcollisional quenching. The intrinsic ﬂuorescence of HSA is mainly
attributable to the single tryptophan residue (Trp-214) located at
the center of its hydrophobic interior adjacent to a hinge region(Sudlow site I) [44]. Compounds that bind to Sudlow site I often
diminish the HSA ﬂuorescence and it was used to investigate theinteraction of drugs with HSA [44e46]. The decrease in the ﬂuorescence intensity is analyzed by the SterneVolmer equation (4):(4)

where F0 and F indicate the steady-state ﬂuorescence intensities in
the absence and in the presence of a quencher (i.e., SRB), respectively, Ksv is the SterneVolmer quenching constant, and [Q] is thequencher concentration. The SterneVolmer plot (Fig. 5) shows
Ksv ¼ 1.1  104 M1, and the apparent quenching rate constant kappqapp
is calculated according to kq ¼ Ksv =s0 , where s0 is the lifetime ofthe ﬂuorophore (i.e., HSA) in the absence of quencher (s0 of HSA isapp
approximately 108 s) [46]. The kq value of 1.1  1012 M1 s1 ishigher than the maximum diffusion-limited rate, implying thatstatic quenching caused by complex formation plays a dominantrole in this quenching process, rather than collisional quenching.
3.4. Circular dichroism
Circular dichroism (CD) spectral measurement is often utilized
to monitor the conformational changes in HSA upon thecomplexation of small molecules [47,48]. The CD spectra of HSA
exhibit a typical shape of an a-helix-rich secondary structure withtwo minima at approximately 208 and 222 nm. The CD spectra
results upon the addition of SRB are expressed as the mean residue
ellipticity (MRE) deﬁned by equation (5):MRE ¼ observed CD=ðc  n  lÞ;(5)where c is the molar concentration of HSA, n is the number ofamino acid residues (585), and l is the path length (1 cm). The
spectral change in Fig. 6 is indicative of SRBeHSA complexation andthe loss of a-helix.
3.5. Energy transfer
The ﬂuorescence and CD spectra studies proved that HSA forms
a complex with SRB. Resonance energy transfer (RET) has beenutilized for measuring molecular distances in biological and

14
12
r/Df × 10−4

13

10
8
12.5

6
ln K

4

12

2

0
0

0.2

0.4

0.6
r

0.8

1

1.2

Fig. 3. Scatchard plot of SRB binding to HSA at 25  C and pH 7.4 (phosphate buffer,
67 mM). r indicates the number of moles of SRB bound per mole of HSA. Df indicatesthe free SRB concentration. The SRB concentrations were varied from 10 to 40 mM atdifferent HSA concentrations (0, 34, or 150 mM). Fluorescence measurements of SRB
were carried out under the following conditions: excitation at 425 nm and emission at
625 nm.

11.5

110.00315

0.00325

0.00335
1/T (K−1)

0.00345

Fig. 4. van’t Hoff plots of the HSAeSRB interaction.

0.00355

M. Kitamura et al. / Dyes and Pigments 99 (2013) 588e593

591

F0 / F

1.3

1.2

1.1

1
0

5

10
15
20
6
[SRB] × 10 (M)25

30
Fig. 7. Spectral overlap between the absorption spectrum of SRB and the ﬂuorescence
spectrum of HSA (25  C and pH 7.4).

Fig. 5. SterneVolmer plot of the ﬂuorescence quenching of HSA (10 mM) upon the
addition of SRB at 25  C and pH 7.4 (phosphate buffer, 67 mM). Excitation and emission
wavelengths were 295 and 340 nm, respectively. F0 and F are the corrected
ﬂuorescence intensities of HSA in the absence and presence of SRB due to theinner-ﬁlter effect, respectively (See Experimental Section).

macromolecular systems [49e52]. RET occurs when the ﬂuorescence emission spectra of a donor (HSA) overlap with the absorption spectra of an acceptor (SRB), as depicted in Fig. 7. By using
equation (6) for RET, the distance r between Trp-214 of HSA and SRB
is calculated.

ZN
J ¼FðlÞεðlÞl dl=
4

ZN

0



E ¼ 1  F=F0 ¼ R60 = R60 þ r 6 ;(6)E denotes the efﬁciency of transfer between the donor and theacceptor, r is the average distance between the donor andacceptor, and R0, known as the Förster distance, is the criticaldistance when the efﬁciency of transfer is 50%. R0 can be obtained
from equation (7):R60 ¼ 8:79  1025  K 2  n4  4  J;

(7)FðlÞdl;(8)0where F(l) is the corrected ﬂuorescence intensity of the donor in
the wavelength range, ε(l) is the molar absorptivity of the acceptor
at the wavelength l. Applying equations (6)e(8), we calculate
R0 ¼ 2.15 nm, E ¼ 0.28, and r ¼ 2.51 nm. The R0 and r values are on
the 2e8 nm scale, and 0.5R0 < r < 1.5R0, indicating the existence ofan interaction between SRB and Trp-214 of HSA.
3.6. Binding site
To study the distinct binding site for SRB on HSA, site-speciﬁc
ligands were used: warfarin and furosemide for site I and

-0.5

-1

1

ΔF/ΔF0

(d)

-1.5
(a)

Warfarin

Furosemide
0.5

Ibuprofen[θ]10−4 (deg·cm 2 /dmol)

K2 is the orientation factor related to the geometry of the donor
and acceptor dipoles, and K2 ¼ 2/3 for random orientation, n is theaverage refractive index of the medium in the wavelength range inwhich spectral overlap is signiﬁcant, 4 is the ﬂuorescence quantum
yield of the donor (0.118 for HSA), and J is the spectral overlap
between the emission spectrum of the donor and the absorption
spectrum of the acceptor, which are calculated using equation (8):

-2

0
-4.5

-2.5
205

210

215

220

225

230

235

Wavelength (nm)Fig. 6. CD spectra of HSA (1 mM) at 25  C and pH 7.4 (phosphate buffer, 67 mM). The
SRB concentrations were (a) 0, (b) 7.8, (c) 15.1, and (d) 22.1. Results are expressed asmean residue ellipticity (MRE) in deg$cm2/dmol.

-4

-3.5
log [Ligand]-3

Fig. 8. Change in ﬂuorescence spectra of SRB (5 mM) bound to HSA (90 mM) upon the
addition of ligand molecules (warfarin, ibuprofen, or furosemide) at 25  C and pH 7.4(phosphate buffer, 67 mM). Excitation and emission wavelengths were 425 and581 nm, respectively. F0 and F are the ﬂuorescence intensities of HSA in the absence
and presence of SRB, respectively.

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M. Kitamura et al. / Dyes and Pigments 99 (2013) 588e593

ibuprofen for site II [53e56]. Fig. 8 shows that the ﬂuorescenceintensity signiﬁcantly decreased after the addition of warfarin andfurosemide but not after adding ibuprofen, suggesting that SRB
binds to site I of HSA. It could be presumed that xanthene moiety of
the SRB is situated in the hydrophobic cavity of the site I and two
sulfonates slightly interact with positively charged residues such as
Lys-195, Lys-199, Arg-218, Arg-222, His-242, and Arg-257 which arelocated at the entrance of the site I cavity.
4. Conclusions
This study presents ﬂuorescence and circular dichroism spectroscopic results of the interaction of SRB with HSA. The increase in
the ﬂuorescence intensity of SRB upon complexation with HSA
revealed the binding constant (1.6  105 M1 at 25  C), binding
stoichiometry (1:1), and thermodynamic parameters of the SRBeHSA interaction (DH ¼ 5.2 kJ/mol, DS ¼ 116.5 J/mol K). The entropydriven binding process (large positive DS and small positive DH)
implies that SRB binds to Sudlow site I of HSA predominantly byhydrophobic interaction. This site-selective binding was conﬁrmed
by site-competitive binding experiments using warfarin and furosemide as marker ligands for site I and ibuprofen for site II. Circulardichroism suggested some degree of conformational changes in
HSA upon binding with SRB. Since HSA is an important biomarker
for clinical practice, SRB could be an alternative to bromocresol
green or bromocresol purple for the measurement of HSA. The
results and data presented here provide important insights into theinteraction of SRB with HSA under physiological conditions, and theuse of SRB should be beneﬁcial in the food coloring industry and indiagnostic imaging.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.dyepig.2013.06.011.
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